The Role of Nitroreductases in Resistance to Nitroimidazoles

biology

Review The Role of Nitroreductases in Resistance to Nitroimidazoles

Carol Thomas 1 and Christopher D. Gwenin 2,*

1 School of Natural Sciences, Bangor University, Bangor LL57 2UW, UK; [email protected] 2 Department of Chemistry, Xi’an Jiaotong-Liverpool University, 111 Ren’ai Road, Suzhou Industrial Park, Suzhou 215123, China * Correspondence: [email protected]; Tel.: +86-(0)512-81888710

Simple Summary: Antimicrobial resistance continues to be a major global health threat. It is estimated by the WHO that 700,000 people die each year because of drug resistance, and this is predicted to rise to 10 million by 2050. As well as the increased cost, which is forecast to exceed $100 trillion, as more expensive drugs have to be deployed, illnesses often last longer and require hospital treatment. This, in turn, increases the strain on often-inadequate healthcare systems. As resistances continue to grow, ﬁnding alternatives is crucial. This review showed that nitroreductases play a role in drug activation but are also associated with resistance mechanisms. These mechanisms require further investigation to fully understand them before they can be utilised against multidrug- resistant organisms. This will depend on committed collaborations between the private and public sector to translate academic research into the clinic.

Abstract: Antimicrobial resistance is a major challenge facing modern medicine, with an estimated 700,000 people dying annually and a global cost in excess of $100 trillion. This has led to an increased need to develop new, effective treatments. This review focuses on nitroimidazoles, which have seen a resurgence in interest due to their broad spectrum of activity against anaerobic Gram-negative and Gram-positive bacteria. The role of nitroreductases is to activate the antimicrobial by reducing the Citation: Thomas, C.; Gwenin, C.D. nitro group. A decrease in the activity of nitroreductases is associated with resistance. This review The Role of Nitroreductases in Mycobacterium tuber- Resistance to Nitroimidazoles. Biology will discuss the resistance mechanisms of different disease organisms, including 2021, 10, 388. https://doi.org/ culosis, Helicobacter pylori and Staphylococcus aureus, and how these impact the effectiveness of speciﬁc 10.3390/biology10050388 nitroimidazoles. Perspectives in the ﬁeld of nitroimidazole drug development are also summarised.

Academic Editor: Keywords: nitroreductases; antimicrobial resistance; mitromidazole; metronidazole; nim genes Vincent Sanchis-Borja

Received: 5 January 2021 Accepted: 10 February 2021 1. Introduction Published: 1 May 2021 Nitroreductases (NTR) are a family of proteins involved in the reduction of nitro- containing compounds [1]. The flexibility of these enzymes comprises their usefulness in a Publisher’s Note: MDPI stays neutral variety of biological and medical applications. These include environmental decontami- with regard to jurisdictional claims in nation using bioremediation [2]; various cancer therapies such as gene and viral-directed published maps and institutional affil- prodrugs [3–7] and probes for detecting hypoxia in tumours [8–11], antiparasitics [12,13], iations. herbicides [14] and for the detection of explosives [15]. For a more detailed review of the many uses of nitroreductases, see the papers by Kumari et al. [11], Zhang et al. [4] and Nepali et al. [16]. Nitroreductases can be divided into two groups: flavin reductases [17] and those from Copyright: © 2021 by the authors. enteric bacteria [18]. Typical NTRs share similar biochemical properties; they usually occur Licensee MDPI, Basel, Switzerland. as a homodimer, contain flavin mononucleotide (FMN) as a cofactor and catalyse using the This article is an open access article ping-pong bi-bi kinetic mechanism [1,19]. The bacterial NTRs can be further split into Type distributed under the terms and I, oxygen-insensitive and type II, which are oxygen-sensitive, as illustrated in Figure1 [20]. conditions of the Creative Commons Bacteria can contain both type I and II, although the most studied are those enzymes that Attribution (CC BY) license (https:// belong to type I [19]. The oxygen-insensitive NTRs can be further divided into major and creativecommons.org/licenses/by/ 4.0/). minor protein groups. The minor group can utilise nicotinamide adenine dinucleotide

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Biology 2021, 10, 388 those enzymes that belong to type I [19]. The oxygen-insensitive NTRs can be further2 ofdi- 16 vided into major and minor protein groups. The minor group can utilise nicotinamide adenine dinucleotide (NAPH) or nicotinamide adenine dinucleotide phosphate (NADPH)(NAPH) or, while nicotinamide the major adenine group dinucleotidecan utilise NADPH phosphate only (NADPH), as the electron while donor the major [21]. group The mostcan utilisestudied NADPH examples only of asthe the major electron and minor donor groups [21]. The are most the E studiedscherichia examples coli NfsA of and the NfsBmajor enzymes and minor [21]. groups are the Escherichia coli NfsA and NfsB enzymes [21].

FigureFigure 1. 1. TheThe reduction reduction scheme scheme of of aa nitro nitro group group and and the the respective respective electron electron transfers transfers required required [2 [200].]. NAD:NAD: nicotinamide nicotinamide adenine dinucleotidedinucleotide and and NADPH: NADPH: nicotinamide nicotinamide adenine adenine dinucleotide dinucleotide phosphate. phosphate. The physiological functions of NTRs are not completely understood, but several have beenThe proposed physiological for oxygen-insensitive functions of NTRs bacterial are not NTRscompletely [19]. Itunderstood has been assumed, but several that have they beenhave proposed a role to for play oxygen in detoxification,-insensitive bacterial as they reduceNTRs [19]. a broad It has range been ofassumed compounds that they [19]. haveSome a arerole involved to play inin specificdetoxification degradation, as they pathways, reduce sucha broad as nitrobenzenerange of compounds reductase [19]. and Somenitrophenol are involved reductase in specific [22,23 ].degradation NTRs may pathways also be involved, such as innit therobenzene response reductase to oxidative and nitrophenolstress, as the reductase enzyme NfsA[22,23]. is NTRs regulated mayby also the be SoxRS involved system, in the which response is involved to oxidative in the stressprevention, as the of enzyme oxidative NfsA damage is regulated [24,25]. Theby the range SoxRS and adaptabilitysystem, which of NTRsis involved have enabled in the preventionsome of these of oxidative enzymes damage to specialise [24,25] in. The different range metabolic and adapta functionsbility of NTRs without have necessarily enabled somelosing of the these other enzymes reductase to specialise activities [in19 ].different Therefore, metabolic while some functions NTRs without are associated necessarily with losingspecific the metabolic other reductase pathways activities or thereduction [19]. Therefore, of different while nitroaromatic some NTRs are compounds, associated others with specificmay be metabolic active in processes pathways such or the as oxidativereduction stressof different response nitroaromatic or bioluminescence compounds, [26]. oth- Due ersto themay ability be active of NTRs in processes to affect thesuch toxic, as oxidative mutagenic stress and carcinogenicresponse or characteristicsbioluminescence of many[26]. Duenitroaromatics, to the ability nitrofuran of NTRs derivativesto affect the have toxic, been mutagenic used to developand carcinogenic a group of characteristics antimicrobials ofcommonly many nitroaromatics, knowns as nitroimidazoles nitrofuran derivatives [27]. have been used to develop a group of an- timicrobialsNitroimidazoles commonly have knowns seen as a nitroimidazoles resurgence in interest [27]. due to their broad spectrum of activity against anaerobic Gram-negative and Gram-positive bacteria. They are particularly Nitroimidazoles have seen a resurgence in interest due to their broad spectrum of being seen as important in the battle against antibiotic resistance. Resistance to antibiotics activity against anaerobic Gram-negative and Gram-positive bacteria. They are particu- is a major challenge facing modern medicine, and its importance was highlighted in 2016 larly being seen as important in the battle against antibiotic resistance. Resistance to anti- when it was a topic for discussion at the United Nations General Assembly [28]. It is biotics is a major challenge facing modern medicine, and its importance was highlighted estimated that 700,000 people die each year because of infections that are resistant to in 2016 when it was a topic for discussion at the United Nations General Assembly [28]. It current antibiotics and that this figure is predicted to rise to 10 million annually by 2050, is estimated that 700,000 people die each year because of infections that are resistant to with the global cost forecast to exceed $100 trillion over the next few decades [29]. The current antibiotics and that this figure is predicted to rise to 10 million annually by 2050, overuse of antibiotics in both clinical and agricultural settings has accelerated the process with the global cost forecast to exceed $100 trillion over the next few decades [29]. The of resistance [29,30]. Perhaps the most widely known antibiotic is penicillin, which was overuse of antibiotics in both clinical and agricultural settings has accelerated the process discovered by Fleming in 1928, although it was not produced on any great scale until of resistance [29,30]. Perhaps the most widely known antibiotic is penicillin, which was 1940 [30]. Most current antimicrobials were discovered between the 1940s and 1970s, discoveredincluding nitroimidazolesby Fleming in 1928 in the, although early 1950s, it was when not produced azomycin on was any isolatedgreat scale from until a crude 1940 [30].extract Most of Streptomycescurrent antimicrobialsbacteria [31 were]. This discovered review will between look at the the 1940s role played and 1970s, by NTRs including in the nitroimidazolesresistance mechanisms in the early of nitroimidazoles. 1950s, when azomycin was isolated from a crude extract of Biology 2021, 10, x 3 of 16

Biology 2021, 10, 388 3 of 16 Streptomyces bacteria [31]. This review will look at the role played by NTRs in the resistance mechanisms of nitroimidazoles. 2. Nitroimidazoles 2. Nitroimidazoles Nitroimidazoles are a class of antimicrobial drugs that have a broad spectrum of Nitroimidazolesactivity against are anaerobica class of antimicrobial Gram-positive drugs and Gram-negativethat have a broad bacteria, spectrum as well of ac- as parasites tivity againstand anaerobic mycobacteria, Gram since-positive their and discovery Gram-negative drugs such bacteria as metronidazole,, as well as parasites pretomanid and and mycobacteriadelamanid, since have their gone discovery on to form drugs a large such part as ofmetronidazole, the treatment pretomanid for Helicobacter and pylori and delamanidMycobacterium have gone on tuberculosis, to form a largerespectively. part of the The treatment mode of action for Helicobacter of nitroimidazoles pylori and can help to Mycobacteriumexplain tuberculosis, why they haverespectively such a broad. The spectrummode of ofaction activity. of nitroimidazoles They are prodrugs can that help require the to explainreduction why they of have the nitrosuch group a broad before spectrum they display of activity. any antimicrobialThey are prodrugs effects. that This re- is normally quire the achievedreduction byof NTRsthe nitro using group flavin before mononucleotide they display any (FMN) antimicrobial or flavin adenineeffects. This dinucleotide is normally(FAD) achieved as prosthetic by NTRs groups using f andlavin either mononucleotide NADH or NADPH (FMN) or as flavin reducing adenine agents di- [27]. The nucleotidemechanism (FAD) as prosthetic is understood groups to have and theeither following NADH steps: or NADPH (i) molecules as reducing enter theagents cells through [27]. The passivemechanism diffusion, is understood (ii) the nitro to have group the is following reduced tosteps reactive: (i) molecules radical species enter t andhe (iii) the cells throughradicals passive react diffusion, with the (ii) DNA the ornitro protein group within is reduced the cell to reactive [27]. The radical reduction species of products and (iii) thewithin radicals a cell react depends with onthe the DNA redox or potentialprotein within of the the compound cell [27]. and The the reduction number of electrons products beingwithin transferred a cell depend [32s]. on The the system redox operatespotential more of the efficiently compoun underd and anaerobicthe number conditions, of electronsmeaning being transferred the bactericidal [32]. The effects system are increased operates comparedmore efficiently to when under oxygen anaerobic is present [33]. conditionsThe, meaning complete the pathway bactericidal can be effects seen inare Figure increased2. The compared diverse mode to when of action, oxygen which is is often present [33inadequately]. The complete defined, pathway can leadcan be to seen problems in Figure when 2. tryingThe diverse to maximise mode of their action, bactericidal which is oftenpotential. inadequately It is also defined, a factor can that lead must to problems be taken intowhen consideration trying to maximise when lookingtheir at the bactericidalimportant potential. issue It is ofalso resistance. a factor that must be taken into consideration when looking at the important issue of resistance.

Figure 2. Mutagenic pathway of nitroarenes [33]. Figure 2. Mutagenic pathway of nitroarenes [33]. ResistanceResistance to nitroimidazoles to nitroimidazoles usually occurs usually because occurs there because is a there decrease is a decrease in the activ- in the activity ity of the ofenzymes the enzymes responsible responsible for the forreduction the reduction of the nitro of the group nitro [34] group. These [34 ].resistance These resistance mechanismsmechanisms differ depending differ depending on the targ onet theorganism target organismand will be and described will be described in more depth in more depth in the sectionsin the below. sections To below. develop To new develop drugs new that drugs will aid that the will management aid the management of infectious of infectious diseases, thesediseases, resistance these resistance mechanisms mechanisms must be better must understood. be better understood. 3. Metronidazole 3. Metronidazole Metronidazole (MTZ) is a nitroimidazole prodrug derived from azomycin, which Metronidazole (MTZ) is a nitroimidazole prodrug derived from azomycin, which has has been used as an antimicrobial since the early 1960s [35]. The drug enters the cells via been used as an antimicrobial since the early 1960s [35]. The drug enters the cells via pas- passive diffusion but is inactive until the nitro group is reduced. This can occur via two sive diffusion but is inactive until the nitro group is reduced. This can occur via two routes routes (Figure3): reductive activation, which results in toxicity, or reductive inactivation, (Figure 3)where: reductive the nitroactivation group, which is reduced results to in a nontoxictoxicity, or amino reductive derivative inactivation [19,36,]. where MTZ is one of the nitro groupthe main is reduced drugs used to a tonontoxic treat Helicobacter amino derivative pylori, which [19,36 is]. MTZ a Gram-negative is one of the microaerophile main drugs usedbacterium to treat Helicobacter that can be pylori found, which in the is a stomach Gram-negative of almost microaerophile 50% of the world’sbacterium population. that can beThe found symptoms in the stomach include of chronic almost gastritis 50% of the and world’s peptic population. ulcers. It is The also symptoms classed as a type I Biology 2021, 10, x 4 of 16 Biology 2021, 10, 388 4 of 16

include chronic gastritis and peptic ulcers. It is also classed as a type I carcinogen by WHO, carcinogenas it is a risk by WHO,factor asin itgastric is a risk adenocarcinoma factor in gastric and adenocarcinoma mucosa-associ andated mucosa-associated lymphoid tissue lymphoidlymphoma tissue (MALT) lymphoma [37–39] (MALT). [37–39].

Figure 3. Mechanism of metronidazole [27] involving bioreduction of the nitro group by ferre- Figure 3. Mechanism of metronidazole [27] involving bioreduction of the nitro group by ferredoxin. doxin. Resistance to MTZ in cases of H. pylori is more common than with other antimicrobials and canResistance be as high to MTZ as 80%, in cases depending of H. pylori on the is more patient common group andthan geographic with other antimicrobi- region [40]. Asals the and mechanism can be as high of action as 80%, requires depending enzymatic on the reduction, patient group anything and geographic which affects region this can[40]. causeAs the resistance. mechanism Mutations of action inrequires the genes enzymatic that encode reduction certain, anything electron which transport affects proteins this can arecause one resistance. such example; Mutations inactivation in the genes of the thatrdxA encode(encodes certain oxygen-insensitive electron transport NADPH proteins nitroreductase)are one such ex andample;frxA inactivation(encodes NADPH of the flavinrdxA (encodes oxidoreductase) oxygen- genesinsensitive is linked NADPH to MTZ ni- resistancetroreductase) in H. and pylori frxA[41 (encodes,42]. Chua NADPH et al. [43 flavin] demonstrated oxidoreductase) a strong genes correlation is linked between to MTZ mutationsresistance thatin H. inactivate pylori [41, RdxA42]. Chua and et its al resistance.. [43] demonstrated They also a reportedstrong correlation that a significant between numbermutations of MTZ-resistant that inactivate strains RdxA of andH. pyloriits resistance.had a mutation They also of the reported Arg-16 residuethat a significant of RdxA. Thisnumber amino of acid MTZ residue-resistant is responsible strains of forH. binding pylori had between a mutation RdxA and of the the ArgFMN-16 phosphoryl residue of group,RdxA. andThis therefore,amino acid any residue mutation is respon maysible adversely for binding affect thebetween reduction RdxA of and MTZ the to FMN its cytotoxicphosphoryl form group [43]., However,and therefore this, cannot any mutation be responsible may adversely on its own affect for conferring the reduction MTZ of resistance,MTZ to its as cytotoxic it has also form been [43] found. However, in H. pylorithis cannotstrains be that responsible were susceptible on its own to MTZfor confer- [43]. Chuaring MTZ et al. [resistance43] proposed, as it that has high also levels been offoundfrxA inwould H. pylori counteract strains thethat effect were of susceptible Arg-16 and to makeMTZ H.[43]. pylori Chuasusceptible et al. [43] toproposed MTZ, despite that high any levels mutation of frxA of rdxA.wouldIf counteractfrxA is inactivated, the effect itof doesArg- not16 and appear make to playH. pylori a major susceptible role in MTZ-resistance to MTZ, despite but any may mutation work together of rdxA. with If frxA other is mutationsinactivated to, increaseit does not the appear resistance to play [43]. a major role in MTZ-resistance but may work to- getherAs with resistance other tomutations MTZ can to occur increase without the resistance either rdxA [43].or frxA inactivation, other mech- anismsAs must resistance be considered to MTZ can [44 ,occur45]. One without such either mechanism rdxA or suggested frxA inactivation by Lee, et other al. [ 46mech-] is aanisms gene, hefAmust, associatedbe considered with [44 the,45] efflux. One pumpsuch mechan HefABC.ism The suggested expression by Lee of et this al. gene[46] is is a increasedgene, hefA when, associated exposed with to MTZ.the efflux In MTZ-resistant pump HefABC. strains The ofexpressionH. pylori ofwith this intact gene frxAis in- andcreasedrdxA when, the levels exposed of hefA to MTZ.were In higher MTZ than-resistant in susceptible strains of strains.H. pylori If withhefA intactwas knocked frxA and out,rdxA the, the strains levels became of hefA sensitivewere higher to MTZ, than andin susceptible this was reversed strains. If by hefA the was addition knocked of hefA, out, showingthe strains that becamehefA was sensitive directly to involvedMTZ, and in this MTZ-resistance was reversed [by46 ].the While addition this couldof hefA, help show- to explain cases of MTZ resistance in the presence of functioning frxA and rdxA genes, the Biology 2021, 10, x 5 of 16

ing that hefA was directly involved in MTZ-resistance [46]. While this could help to ex- Biologyplain2021, 10 ,cases 388 of MTZ resistance in the presence of functioning frxA and rdxA genes, the 5 of 16 overexpression of hefA alone does not result in resistance [46]. Clearly, the resistance of H. pylori to MTZ is a complex mechanism that requires further study to fully understand. Nitroimidazole overexpressionresistance at oflowhefA levelsalone is does often not resultassociated in resistance with [the46]. Clearly,nim genes the resistance[47]. of The Nim genes were H.first pylori describedto MTZ is in a complex1994 [48] mechanism, and their that nitroreductase requires further studyactivity to fully was understand. ob- Nitroimidazole resistance at low levels is often associated with the nim genes [47]. The served two years laterNim whengenes a werenimA first-positive described strain in 1994 of [Bacteroides48], and their fragilis nitroreductase reduced activity a nitroim- was observed idazole drug to its noncytotoxictwo years later amine when a derivativenimA-positive [49 strain]. Currently, of Bacteroides eleven fragilis nimreduced genes a nitroimidazole have been identified: nimAdrug–nimK to its [50] noncytotoxic. amine derivative [49]. Currently, eleven nim genes have been The exact mechanismidentified: of nimAnim resistance–nimK [50]. is not fully understood, and there is conflict- The exact mechanism of nim resistance is not fully understood, and there is conflicting ing evidence for the roleevidence of nim for the genes role ofinnim metronidazolegenes in metronidazole (MTZ) (MTZ)resistance resistance [50, [5150,].51 Figure]. Figure 4 below below shows the involvementshows the involvement of nim genes of nim in genesthe mechanism in the mechanism and resistance and resistance of ofmetroni- metronidazole. dazole. The overexpressionThe overexpression of nimA, nimE of nimA and, nimE nimJand inducesnimJ induces an increase an increase in in MTZ MTZ resistance resistance in E. coli in E. coli and B. fragilisand, respectivelyB. fragilis, respectively [52,53]. [ 52,53].

FigureFigure 4. 4.Summarised Summarised mode mode of action of action and main and mechanisms main mechanisms involved in involved resistance. (in1) resistance. Shows nitroimidazole (1) Shows reductase activitynitroimidazole encoded by reductase the nim genes, activity (2) metabolic encoded shift by away the tonim the genes pathway, (2 related) metabolic to conversion shift away of pyruvate to the to path- lactate via lactateway related dehydrogenase to conversion (3) increased of pyruvate efﬂux of the to antibiotic lactate via (4) increasedlactate dehydrogenase DNA repair capacity (3) (increased5) activation efflux of antioxidant of defensethe antibiotic systems ( (46)) increased deﬁciency ofDNA the ferrous repair ironcapacity transporter (5) activation FeoAB, (7 )of overexpression antioxidant ofdefense the rhamnose systems catabolism (6) regulatorydeficiency protein of the RhaR ferrous [50]. iron transporter FeoAB, (7) overexpression of the rhamnose catabolism regulatory protein RhaR [50]. However, several Bacteroides spp. and genera within the Clostridia class are resistant to MTZ without containing the nim gene, while other nim-positive species are MTZ- However, severalsusceptible Bacteroides [54– spp.58]. Thus,and genera the presence within of athenim Clostridiagene does notclass automatically are resistant equal re- to MTZ without containingsistance. the Gal nim et al. gene, [54] found while that other only nim about-positive half of the spec 50 iesBacteroides are MTZstrains-sus- positive ceptible [54–58]. Thus,for the the presencenim gene presented of a nim minimumgene does inhibitory not automatically concentrations equal (MICs) resistance. ranging from 16 to >32 µg/mL—therefore, above the resistance breakpoint. Sethi et al. [59] looked at the Gal et al. [54] found MTZthat resistanceonly about rates half in India of the and 50 the Bacteroides relationship strains of isolates positive positive for for athenim nimgene with gene presented minimumresistance. inhibitory They found a concentrations relatively high rate (MICs of resistance) ranging (31% compared from 16 to <1% to >32in Europe), µg/mL—therefore, abovewith a 53%the rateresistance of positivity breakpoint. for the nim Sethigene. Outet al. of 20[59] samples, looked 12 thatat the were MTZ positive for resistance rates in India and the relationship of isolates positive for a nim gene with resistance. They found a relatively high rate of resistance (31% compared to <1% in Europe), with a 53% rate of positivity for the nim gene. Out of 20 samples, 12 that were positive for the nim gene also showed resistance to MTZ, a significant correlation [59]. Leitsch et al. [60] ascertained the levels of expression of the nim genes using 2D gel electrophoresis Biology 2021, 10, x 6 of 16

(2DE). The study looked at whether adaptation to an increased concentration of MTZ led to increased levels of the nim protein. As nim proteins are thought to act as nitroreductases that reduce MTZ to its noncytotoxic form, their profusion should correlate with high levels of MTZ resistance. To test this theory, a high-level MTZ resistance was induced in three strains of B. fragilis, but when the nim levels were measured, there was found to be no upregulation [60]. Such studies reinforce the complexity of MTZ resistance mechanisms and emphasise that nim genes alone are not sufficient to give high-level MTZ resistance Biology 2021, 10, 388 6 of 16 [53,54,56]. Nim genes have been found in multidrug resistant strains of B. fragilis, together with other resistance genes [61]. A cluster of multidrug-resistant (MDR) B. fragilis isolates thecontainingnim gene also showednimB, resistancecfiA, ermF to MTZ, and a significanttetQ genes correlation was [detected59]. Leitsch etusing al. [60] whole-genome sequencing ascertained(WGS) the[61] levels. Nim of expression genes ofhave the nim alsogenes been using 2Dfound gel electrophoresis in an MDR (2DE). strain The of B. thetaiotaomicron to- study looked at whether adaptation to an increased concentration of MTZ led to increased levelsgether of the withnim protein. two Asβ-nimlactamaseproteins aregenes, thought two to act tetX as nitroreductases genes—tetQ that reduce and ermF—two cat genes and MTZseveral to its noncytotoxic genes encoding form, their efflux profusion pumps should correlate [62]. Despite with high levelsthis ofcurrent MTZ inability to definitively resistance. To test this theory, a high-level MTZ resistance was induced in three strains of B. fragilisdemons, but whentrate the thenim levels cause were and measured, effect there of was nim found genes to be noand upregulation MTZ resistance [60]. , it is still worthy of Suchfurther studies investigation reinforce the complexity as important of MTZ resistance risk mechanismsfactors, such and emphasise as the thatlocation of the nim genes on nim genes alone are not sufficient to give high-level MTZ resistance [53,54,56]. Nim genes havemobile been found genetic in multidrug material resistants plus strains the of highB. fragilis usage, together of withMTZ other as resistancea frontline drug in multiple infec- genestious [61 ].diseases A cluster of could multidrug-resistant lead to an (MDR) increaseB. fragilis inisolates resistance containing [50].nimB This, cfiA, is underlined by the pres- ermFenceand oftetQ nimgenes genes was detected in several using whole-genome MDR anaerobes sequencing [63] (WGS). [61]. Nim genes have also been found in an MDR strain of B. thetaiotaomicron together with two β-lactamase genes, two tetX genes—tetQ and ermF—two cat genes and several genes encoding efflux pumps4. Delamanid [62]. Despite this and current Pretomanid inability to definitively demonstrate the cause and effect of nim genes and MTZ resistance, it is still worthy of further investigation as important risk factors,Delamanid such as the locationand pretomanid of the nim genes are on both mobile bicyclic genetic materials 4-nitroimidazoles plus the , and phase II clinical hightrials usage have of MTZ shown as a frontline them drugto be in effective multiple infectious against diseases both couldreplicating lead to an and hypoxic nonreplicating increase in resistance [50]. This is underlined by the presence of nim genes in several MDR anaerobesmycobacteria [63]. [64]. Tuberculosis (TB) is the leading cause of death from infectious disease.

4.In Delamanid 2018, 10 and million Pretomanid people became ill with TB, with 1.2 million deaths [65]. Under aerobic conditionsDelamanid and, the pretomanid mechanism are both of bicyclic action 4-nitroimidazoles, is to inhibit andthe phase formation II clinical of mycolic acids [66], while trialsunder have shown anaerobic them to be conditions effective against, the both mechanism replicating and hypoxic induces nonreplicating respiratory poisoning [67]. Preto- mycobacteria [64]. Tuberculosis (TB) is the leading cause of death from infectious disease. Inmanid, 2018, 10 million in particular, people became donates ill with TB, nitric with 1.2oxide million (NO) deaths, which [65]. Under can aerobic create toxic conditions within conditions,the bacilli the in mechanism nonreplicating of action is MTB to inhibit [67 the]. Both formation delamanid of mycolic and acids pretomanid [66], are prodrugs that whilerequire under bioactivationanaerobic conditions, of the the mechanism nitro group induces to respiratory become poisoning effective. [67]. They are activated in an Pretomanid, in particular, donates nitric oxide (NO), which can create toxic conditions withinF420H2 the bacilli-dependant in nonreplicating reaction MTB [67 ]. in Both MTB delamanid by and deazaflavin pretomanid are-dep prodrugsendant nitroreductase (Ddn) that[67 require,68]. bioactivationTherefore, of mutations the nitro group that to become affect effective. the activity They are activatedof Ddn in or an the biosynthesis or reduc- F420H2-dependant reaction in MTB by deazaﬂavin-dependant nitroreductase (Ddn) [67,68]. Therefore,tion of mutations F420 may that affectresult the in activity resistance of Ddn or the[64 biosynthesis]. or reduction of F420 may resultDelamanid in resistance [(Figure64]. 5) was discovered by Otsuka Pharmaceuticals [69] and was granted Delamanid (Figure5) was discovered by Otsuka Pharmaceuticals [ 69] and was granted conditionalconditional approval approval in 2014 by in the 2014 European by Medicinesthe European Agency (EMA) Medicines for the treatment Agency (EMA) for the treatment ofof pulmonary pulmonary MDR-TB MDR in adults-TB in in combination adults in with combination other frontline with drugs [other70]. frontline drugs [70].

FigureFigure 5. Structure 5. Structure of delamanid of d [69elamanid]. [69]. The World Health Organisation (WHO) issued a policy guidance, which stated that delamanidThe could World be added Health to the Organisation WHO-recommended (WHO) regimen issued [71]. It a has policy a potent guidance, which stated that minimum inhibitory concentration (MIC) range of 6–240 ng/mL, which is the lowest among thedelamanid current TB drugs could [69, 72be]; plus,added it showed to the no WHO signs of-recommended toxicity in the Ames regimen test [73]. [71]. It has a potent min- Inimum a phase inhibitory IIa trial, delamanid concentration showed good (MIC) activity range in drug-susceptible of 6–240 TBng/mL patients,, which is the lowest among but due to poor adsorption at high doses, twice-daily dosing was required (2 × 50-mg tabletsthe current per dose) [TB74,75 drugs]. The compound [69,72]; worksplus, by it inhibitingshowed the no synthesis signs ofof cell toxicity wall in the Ames test [73]. In componentsa phase IIa and trial, is active delamanid against replicating showed and nonreplicating good activity persister in drug bacteria,-susceptible both TB patients, but due extrato poor and intracellular adsorption bacilli [at69 ].high It does doses, not show twice a cross-resistance-daily dosing with other was TB drugsrequired (2 × 50-mg tablets per and can therefore be used in combinations [76]. A phase II trial was completed in 2012 dose) [74,75]. The compound works by inhibiting the synthesis of cell wall components and is active against replicating and nonreplicating persister bacteria, both extra and intracellular bacilli [69]. It does not show a cross-resistance with other TB drugs and can therefore be used in combinations [76]. A phase II trial was completed in 2012 in combination with an optimised background regime (OBR) in 17 centres across nine countries Biology 2021, 10, x 7 of 16

[77]. Phase III trials have since been completed, and further investigations into its use in children are underway [77]. Dormant bacteria are often resistant to treatment, and therefore, new drugs with the ability to kill dormant bacilli are more likely to be effective against MDR-TB. The Wayne model is being used to test the efficacy of TB drugs under low oxygen concentrations [78,79]. Various studies have used a modified version of this model to investigate the bactericidal effects of delamanid in aerobic versus anaerobic settings, with Upton et al. [80,81] Biology 2021, 10, 388 showing that it killed 99% of MTB bacilli at 4.4 µg/mL. Delamanid7 of was 16 also as effective as

Biology 2021, 10, x the frontline drug rifampicin against intracellular mycobacteria7 of 16 [82].

A resistance to delamanid has been reported in clinical isolates of TB. Bacilli resistant in combination with an optimised background regime (OBR) in 17 centres across nine countriesto delamanid [77]. Phase have III trials mutations have since in been one completed, of the five and genes further (Figure investigations 6) associate into d with the F420- [77]. Phaseitsdependant use III intrials children have nitroreduction since are underway been completed [77 pathway]. , and further: fgd1, investigations ddn, fbiA, intofbiB its and use infbiC [83]. FbiA, fbiB and fbiC children are underway [77]. are Dormantcoded bacteriafor proteins are often FbiA, resistant FbiB to treatment, and FbiC and, therefore,which neware drugsessential with thefor the biosynthesis of Doabilityrmant bacteria to kill dormant are often bacilli resistant are more to treatment likely to, and be effective therefore against, new drugs MDR-TB. with The the Wayne ability tomodelF420 kill dormant, is with being usedeachbacilli to geneare test more the affecting efficacy likely ofto TBbea differenteffective drugs under against stage low oxygenMDR [84-TB.,85 concentrations ]The; fgd1 Wayne (gloucose [78,79]. -6-phospahte dehy- model isVariousdrogenase) being studiesused to haveplay test usedthe a roleefficacy a modified in ofthe TB version F420drugs of redoxunder this model low recycling oxygen to investigate concentration mechanism the bactericidals and ddn is the reductase [78,79]. effectsVariousrequired of studies delamanid for haveactivation in used aerobic a modified versus[86]. anaerobic version of settings, this model with to Upton investigate et al. [the80, 81bac-] showing tericidalthat effect its killed of delamanid 99% of MTBin aerobic bacilli versus at 4.4 anaerobicµg/mL. Delamanidsettings, with was Upton also et as al. effective [80,81] as the showingfrontline that it killed drug 99% rifampicin of MTB against bacilli intracellularat 4.4 µg/mL. mycobacteria Delamanid was [82 ].also as effective as the frontlineA drug resistance rifampicin to delamanid against intracellular has been reported mycobacteria in clinical [82] isolates. of TB. Bacilli resistant A rtoesistance delamanid to delamanid have mutations has been in onereported of the in five clinical genes isolates (Figure of6 )TB. associated Bacilli resistant with the F420- to delamaniddependant have mutations nitroreduction in one pathway: of the fivefgd1, genes ddn, (Figure fbiA, 6) fbiB associateand fbiCd with[83]. theFbiA F420, fbiB- and dependantfbiC nitroreductionare coded for proteinspathway FbiA,: fgd1, FbiB ddn, andfbiA, FbiC, fbiB and which fbiC are [83 essential]. FbiA, fbiB for theand biosynthesis fbiC are codedof F420,for proteins with each FbiA, gene FbiB affecting and FbiC a, different which are stage essential [84,85 ];forfgd1 the (gloucose-6-phospahtebiosynthesis of F420, withdehydrogenase) each gene affecting play a rolea different in the F420 stage redox [84,85 recycling]; fgd1 (gloucose mechanism-6-phospahte and ddn is dehy- the reductase drogenase)required play fora role activation in the F420 [86]. redox recycling mechanism and ddn is the reductase required for activation [86].

Figure 6. Gene products involved in the bioactivation of delamanid [86].

Fujiwara et al. [86] showed that, from 30 randomly selected resistant colonies, the frequency of mutations across the five genes varied considerably: ddn (20%), fgd1 (30%), fbiA (16.7%), fbiB (6.7%) and fbiC (26.7%). The fact that resistant bacilli lack the ability to Figure 6.Figureactivate Gene products 6. Gene delamanid products involved involved in theis bioactivationshown in the bioactivation by of thedelamanid following; of delamanid [86]. [ 86when]. drug-susceptible MTB bacilli were incubated with delamanid, the concentration decreased as it was converted to the des- FujiwaraFujiwara et al. [86 et] al.showed [86] showed that, from that, 30 from randomly 30 randomly selected selected resistant resistant colonies colonies,, the the frequencyfrequencynitroimidazole of mutations of mutations across form acrossthe ,five but the genes fivewhen genes varied resistant varied considerably considerably: bacilli: ddn were ddn(20%),(20%), used fgd1fgd1 ,(30%), no(30%), change fbiA was seen [82,86]. fbiA (16.7%),(16.7%), fbiBPretomanidfbiB (6.7%)(6.7%) and and fbiC (FigurefbiC (26.7%).(26.7%). 7) The was The fact fact identified that that resistant resistant by bacilli bacilli PathoGenesis lack thethe abilityability and toto activate was approved by the FDA delamanid is shown by the following; when drug-susceptible MTB bacilli were incubated activate indelamanid 2019 for is shownthe treatment by the following; of multidrug when drug--resistantsusceptible TMTBB in bacilli combination were with bedaquilne and incubatedwith with delamanid, delamanid the, concentrationthe concentration decreased decreased as it wasas it converted was converted to the des-nitroimidazoleto the des- form, but when resistant bacilli were used, no change was seen [82,86]. nitroimidazolelinezolid form [, 87but]. when The resistantMIC values bacilli were range used from, no change 150 to was 250 seen ng/mL [82,86]., and no cross-resistance with Pretomanid (Figure7) was identified by PathoGenesis and was approved by the FDA Pretomanidother TB (Figure drugs 7) was has identified yet been by PathoGenesis seen, making and was it a approved good candidate by the FDA for treating MDR-TB [88]. in 2019 for the treatment of multidrug-resistant TB in combination with bedaquilne and in 2019 for the treatment of multidrug-resistant TB in combination with bedaquilne and linezolidStudies [87 have]. The MIC shown values that range pretomanidfrom 150 to 250, ng/mL, in combination and no cross-resistance with moxifloxacin with and pyra- linezolid [87]. The MIC values range from 150 to 250 ng/mL, and no cross-resistance with other TB drugs has yet been seen, making it a good candidate for treating MDR-TB [88]. other TBzinamide drugs has yet, had been better seen, making bactericidal it a good activitycandidate thanfor treating the current MDR-TB standard[88]. regimen [89,90]. The Studies have shown that pretomanid, in combination with moxifloxacin and pyrazinamide, Studies primary have shown mechanism that pretomanid of action, in combination is the inhibition with moxifloxacin of synthesis and pyra-of cell wall lipids and proteins had better bactericidal activity than the current standard regimen [89,90]. The primary zinamide, had better bactericidal activity than the current standard regimen [89,90]. The mechanism[91]. of action is the inhibition of synthesis of cell wall lipids and proteins [91]. primary mechanism of action is the inhibition of synthesis of cell wall lipids and proteins [91].

Figure 7.Figure Structure 7. Structure of pretomanid of pretomanid [91]. [91]. Figure 7. Structure of pretomanid [91]. Resistant mutants have shown that both Ddn and FGD1 are essential for the drug to become activatedResistant; however, mutants only Ddn haveis involved shown in reducing that both the nitro Ddn group, and producing FGD1 are essential for the drug to three primarybecome metabolites activated and ;reactive however, nitrogen only species Ddn [67 is]. involvedA study by Haverin reducing et al. [88 ]the nitro group, producing three primary metabolites and reactive nitrogen species [67]. A study by Haver et al. [88] Biology 2021, 10, 388 8 of 16

Resistant mutants have shown that both Ddn and FGD1 are essential for the drug to become activated; however, only Ddn is involved in reducing the nitro group, producing three primary metabolites and reactive nitrogen species [67]. A study by Haver et al. [88] looked at 183 spontaneous pretomanid-resistant mutations of which 29% of the lesions were seen in ddn, 26% in fbiC, 19% in fbiA, 7% in fgd1 and 2% in fbiB; the remaining 17% did not show mutations in any of the five genes. Eighty-three percent had single mutations in one of the genes. As 17% had no mutations within the five genes, it can be hypothesised that other targets are involved in either the mechanism of action or activation pathway of pretomanid [88]. As stated previously, mutations that knock out the activity of Ddn completely may result in resistance in both delamanid and pretomanid. However, this could affect the fitness of MTB, as the cofactor F420 has been shown to be essential for the survival of MTB, used by at least 28 enzymes [92] that play important roles in the hypoxic recovery and evasion of the hosts immune system [93,94]. Therefore, in order for nitroimidazole resistance to spread, the native activity of Ddn must either be retained or compensated for. The activation of delamanid and pretomanid by Ddn is a promiscuous activity that is more prone to mutation than native functions [95], meaning that the prodrug activation could be lost without loss of the native function. Lee et al. [96] conducted studies to better understand the fitness costs of the loss of Ddn activity. They were able to demonstrate, through studying 75 mutants, that a number were able to prevent the activation of pretomanid without the loss of all native functions. Therefore, these mutants would be fit enough to spread to new patients [96]. These mutations are likely to be one of the main routes through which resistance to these two new TB drugs will spread. Of the 75 mutants studied, 25 did not reduce pretomanid, but only 10 lost the ability to reduce delamanid, despite their similarities, suggesting that they interact with Ddn in different ways [96]. Clearly, the results from this study should be considered when looking at the continued development and clinical use of nitroimidazoles. Both delamanid and pretomanid are undergoing phase II/III trials, in combination with other drugs, against drug-susceptible TB and MDR-TB. Mutations of Ddn should be closely monitored to ensure that the best drug combination is used, and that will ultimately reduce the spread of resistance. The indication that delamanid binds to Ddn differently than pretomanid means that it could be used to treat some TB strains that are resistant to pretomanid [96]. A combination of the two could also be used to reduce the spread of resistance. Further testing of a range of nitroimidazoles against a variety of Ddn variants could help to identify drugs which are less likely to develop resistance. To fully understand the resistance mechanism, Fujiwara et al. [86] suggested that a rapid drug susceptibility test should be developed. Presently, only phenotypic tests are available for delamanid based on cultures, with results taking several weeks [97]. However, the development of a molecular based test has been problematic due to the distribution of mutations across five genes [86]. One reason why drugs containing a nitro group have not been more widely used is because of the associations with toxicity, including bone marrow suppression, hepatotoxicity and carcinogenicity [16]. Both types of nitroreduction (I and II) produce toxicity; in type I, this is as a result of the generation of hydroxylamine, and during type II, the generation of reactive oxygen species (ROS) via redox cycling causes pharmacological activity [98]. Therefore, if they are going to play any part in the fight against drug resistance, any new developments must aim to reduce these issues. Drugs such as delamanid and pretomanid have been shown to not be mutagenic or genotoxic, but their use is hampered by issues with solubility. This is an area that could be further investigated by formulation chemists [16].

5. Chloramphenicol The ever-increasing resistance to modern antibiotics has led to the re-evaluation of drugs that had either limited use or were discarded due to toxicity or efﬁcacy issues. One such antimicrobial that is now being reassessed is chloramphenicol [99,100]. Chlorampheni- Biology 2021, 10, 388 9 of 16

col can cross the blood–brain barrier and other sites that can be hard to target, which makes it a powerful drug in the treatment of bacterial meningitis [99]. However, chloramphenicol has also been linked with haematological toxicity [101], and its use is carefully controlled in developed countries [101]. As a possible consequence of this, it can still be effective against multidrug-resistant organisms, including MRSA [102,103]. The main mechanisms of chloramphenicol resistance occur through functions such as enzymatic inactivation via chloramphenicol acetyltransferance, efflux pump removal and ribosome protection [104]. The bacterial modification of chloramphenicol was first reported only two years after it was discovered. Several species of bacteria were reported as being able to reduce the nitro group, with the resulting compound having no antimicrobial effect [104]. Crofts et al. [104] set out to identify the bacterial genes that could be involved in this reduction mechanism. They chose to focus on type I oxygen-insensitive nitroreductases—in particular, NfsB from Haemophilus influenzae. They determined the ability of H. influenzae NfsB to reduce the nitro group of chloramphenicol completely to an amine. The reduction is enough to confer resistance to chloramphenicol, but Crofts et al. observed that the H. influenzae NfsB enzyme also uses metronidazole (MTZ) as a substrate but does not completely reduce the nitro group. They hypothesised that, while NfsB protects the cell against chloramphenicol, it activates MTZ to its cytotoxic state, therefore offsetting any survival benefits. They concluded that the two drugs could be used together to combat bacteria resistant to chloramphenicol on its own [104]. One of the reasons that chloramphenicol has not been more widely used is the concern that its use could result in the development of aplastic anaemia [105]. The mechanism through which this happens is not fully understood, although it is thought that nitro reduction is involved. The identification of a bacterial enzyme that has been shown to reduce chloramphenicol means that this theory can now be tested and hopefully identify analogues of chloramphenicol that can be used in the clinic [104].

6. MT02 The antimicrobial drug MT02 has been identified as a new candidate that is active against Gram-positive bacteria such as Staphylococcus aureus [106]. S. aureus is a common pathogen associated with a wide range of infections, both superficial and invasive [107]. Together with S. epidermidis, it accounts for more than 20% of all infections associated with hospitalisation, affecting over 250,000 patients in the USA and Europe annually [107]. The first antibiotic used to treat S. aureus was penicillin; prior to this, the infection was normally fatal [108]. Resistant strains soon appeared, which, in turn, stimulated the search for new antibiotics in the 1950s, such as streptomycin and tetracycline, but resistance soon developed once they were being routinely used in the clinic [109,110]. The development of methicillin was a result of the search for an antibiotic that would be active against penicillin- resistant S. aureus. Methicillin-resistant S. aureus strains (MRSA) emerged in the 1960s, rendering the drug ineffective [111]. MT02 is a nitro-active compound, and the work carried out by Menzel et al. [106] in 2011 showed that it is a DNA-binding compound leading to the inhibition of DNA replication. It shows high levels of antimicrobial activity against S. aureus and other Gram-positive bacteria [106]. El-Hossary et al. [112] carried out further work to identify the potential of S. aureus to develop resistance to MT02 [112]. In order to select MT02-resistant clones, S. aureus strain MA12 was cultivated with increasing concentrations of MT02. Once the concentration reached 10 µM, a red colour began to appear, which then intensified with increasing concentrations. Work to identify the structure of this red compound showed the stepwise reduction of the aromatic nitro groups of MT02 to amino groups. The conclusion was that this reduction was induced by an enzyme found in the resistant S. aureus strain [112]. Four previously identified enzymes with nitroreductase activity (SAUSA300_0788, SAUSA300_0381, SAUSA300_1986 and SAUSA300_2462) were found not to be responsible for this reduction of MT02 [112]. The group carried out total RNA sequencing (RNA-seq), which identified the overexpression of an assumed nitroreductase SAUSA300_0859. This was confirmed by RT-PCR, which showed a 160- Biology 2021, 10, 388 10 of 16

fold overexpression of SAUSA300_0859 in the MT02-resistant strain. The importance of SAUSA300_0859 in the development of resistance to MT02 was conﬁrmed when sensitivity was restored by a transposon insertion mutant. A biochemical analysis showed that the SAUSA300_0859-encoded protein produced an intense red colour when incubated with MT02, demonstrating that it is the functional enzyme giving resistance in S. aureus [112]. While the molecular mechanism that leads to the overexpression of SAUSA300_0859 is not known, this is thought to be the ﬁrst example of a nitroreductase-based antibiotic resistance mechanism in S. aureus.

7. Future Perspectives As resistances to antimicrobials continue to grow, finding alternatives is crucial. As this review demonstrated, nitroimidazoles have a broad range of activity against many different organisms, and it would therefore be prudent to re-examine their potential. Several strategies that merit further investigation are briefly discussed below. As well as looking for ways to combat the issue of toxicity, the mechanism of nitroimidazole resistance must be fully understood if effective drugs are to be developed. What is clear from the discussions above is that the role played by nitroreductases needs further investigation. This is of particular importance, as many bacteria have developed resistance to multiple drugs [113]. Strategies that aim to directly combat different resistance mechanisms are more likely to be effective in finding ways to treat drug-resistant organisms. The repurposing of existing antimicrobials and their use in novel combinations is one such strategy. Drug repurposing is defined as “an approved drug in one disease area is found to be active in another disease whereas drug repositioning is the uses of a drug active in one disease as a template for the synthesis of derivatives active in another disease” [114]. Drug rescue is defined as “developing new uses of a drug that failed to progress through clinical studies or which was removed from the market” [114]. All these strategies have been applied to nitroimidazoles. Collaboration is important in drug development and repurposing. Open databases provide cost-effective access to the sharing of resources and data. Examples include Pub- Chem and Chemspider, which contain chemical structural information, while DrugBank and SuperTarget provide profiles of drugs and target diseases [115]. As NTRs have such a broad spectrum of activity, the use of these databases presents a useful tool. For example, entering “nitroimidazole” as a search in PubChem (December 8th, 2020) identified 3479 molecules in the compound database and 1754 records in the Bioassay database. Research groups can use these databases to profile nitroimidazoles against a much greater number of microorganisms than would have previously been possible. This could poten- tially identify those with untapped potential. Along similar lines, the open innovation concepts, such as the Community for Open Antimicrobial Drug Discovery (CO-ADD) funded by the Wellcome Trust and The University of Queensland, encourage the open sharing of data and ideas. These initiatives aim to remove any barriers that could limit an individual group’s ability to develop suitable compounds by making resources widely available. This is particularly relevant to NTRs, as their complex modes of action require extensive investigation to fully understand [27]. As previously discussed, NTRs can be used as prodrug activators. Celik et al., [116] investigated the potential for NTRs to be used in antibiotic activation. They aimed to identify possibilities for redesigning existing drugs as precursor prodrugs that could then be activated by bacterial NTRs. They redesigned sulfamethoxazole as a prodrug, and when this was activated by a type I NTR, the results showed an improved antimicrobial action, thus demonstrating that the use of existing approved antibiotics in the form of pre-antibiotics could be used against disease-causing bacteria [116]. The use of antibiotic adjuvants is another strategy that has been shown to work [117]. These are small molecules that, although they have no antimicrobial actions, can increase the efficiency of antibiotics [114]. Biology 2021, 10, 388 11 of 16

The development of more robust vaccines for TB would see the need for aggressive treatments with antibiotics reduced. One such area of research is the use of latency- associated antigens, particularly those encoded by the dormancy survival regulator (DosR) regulon [118]. The current BCG vaccine offers poor protection against adult TB [119], as it does not protect against the bacteria when they are in the dormant phase. As most TB patients have a latent infection before any clinical signs are present, latency-associated antigens that are expressed during the dormant phase are of interest as potential vaccine candidates. Kwon et al. [120] selected Rv3131, which is a hypothetical NTR with two DosR-binding sites, as a vaccine candidate. They aimed to investigate whether Rv3131 had any potential as a vaccine against TB. They concluded that it showed promise, as it met the following criteria: it was recognised by the immune system in in vivo experiments, could induce Ag-speciﬁc Th-1 T cells and showed activity against highly virulent TB strains [120].

8. Conclusions This review has shown that nitroreductases play an important role in drug activation but are also associated with resistance mechanisms. This review highlighted the urgent need for further investigations to fully understand these mechanisms before they can be utilised against multidrug-resistant organisms for the development of new drugs. This will depend on the committed collaborations between the private and public sectors to translate academic research into the clinic.

Author Contributions: All authors contributed equally to conception C.T. drafted the review and C.D.G. provided the funding a proof read. Both authors approved the version to be published, and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors have read and agreed to the published version of the manuscript. Funding: The authors would like to acknowledge the Celtic Advanced Life Sciences Network (CALIN) which is supported by the European Regional Development Fund through the Ireland Wales Cooperation programme. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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